Epitopes or antigenic determinants of a protein antigen represent the sites that are recognized as binding sites by certain immunoglobulin molecules known as antibodies. While epitopes are defined only in a functional sense i.e. by their ability to bind to antibodies, it is accepted that there is a structural basis for their immunological reactivity.
Epitopes are classified as continuous and discontinuous (Atassi and Smith, 1978, Immunochemisty, vol 15 p. 609). Discontinuous epitopes are composed of sequences of amino acids throughout an antigen and rely on the tertiary structure or folding of the protein to bring the sequence together. In contrast, continuous epitopes are short linear peptide fragments of the antigen that are able to bind to antibodies raised against the intact antigen.
Many antigens have been studied as possible serum markers for different types of cancer because the serum concentration of the specific antigen may be an indication of the cancer stage in an untreated person. As such, it would be very advantageous to develop immunological reagents that react with the antigen, and more specifically, with the epitopes of the protein antigen.
To date, methods using physical-chemical scales have attempted to determine the location of probable peptide epitopes which includes looking at the primary structure, that being the amino acid sequence, secondary structure such as turns, helices, and even the folding of the protein in the tertiary structure. Continuous epitopes are structurally less complicated and therefore may be easier to locate, however, the ability to predict the location, length and potency of the site is limited.
Various methods have been used to identify and predict the location of continuous epitopes in proteins by analyzing certain features of their primary structure. For example, parameters such as hydrophilicity, accessibility, and mobility of short segments of polypeptide chains have been correlated with the location of epitopes (see Pellequer et al. 1991, Method in Enzymology, vol 203, p. 176-201).
Hydrophilicity, has been used as the basis for determining protein epitopes by analyzing an amino acid sequence in order to find the point of greatest local hydrophilicity as disclosed in U.S. Patent No. 4,554,101. Hopp and Woods (See Proc. Natl. Acad. Sci. USA, vol. 78, No. 6, pp. 3824-3828, June 1981) have shown that by assigning each amino acid a relative hydrophilicity numerical value and then averaging local hydrophilicity that the highest local average hydrophilicity value is located in or immediately adjacent to the epitope. However, this method does not provide any information as to the optimal length.
Likewise, the amino acid sequence of a protein as measured by the Kyte-Doolittle (Kyte and Doolittle, 1982, J. Mol. Biol. vol. 72, p. 105) scale, is commonly used to evaluate the hydrophilic and hydrophobic tendencies of polypeptide chains by using a hydropathy scale. Each amino acid in the polypeptide chain is assigned a value reflecting its relative hydrophilicity and hydrophobicity which are averaged across a moving section of the sequence. This method offers a graphic visualization of the hydropathic character of the amino acid chain. By using the hydropathic character of the sequence, interior sequence regions which are usually composed of hydrophobic amino acids can be distinguished from hydrophilic exterior sequence regions. This information offers the ability to evaluate the possible secondary structure. However this model, does not predict the optimal length of the epitope or indicate if the effective size of epitopes is unique for each protein molecule.
Accordingly, what is needed is a simple method to identify a peptide epitope and determine the optimal length, location of the epitope within a polypeptide and determine its level of immunopotency.